Label-free quantitative proteomics analysis showed that proteins involved in the lipid metabolism, sirtuin signaling, mitochondrial function, cytoskeletal organization, and antioxidant defense were the most affected. A histopathological analysis elucidated large foci of fibrotic tissue, myocardial lipomatosis and lymphocytic infiltrations in the irradiated samples. These data highlight the suitability of FFPE material for proteomics analysis. The study confirms the previous results emphasizing the role of adverse metabolic changes in the radiation-associated IHD. Most importantly, it excludes age at the time of death as a confounding factor.
Epidemiological studies in the Mayak Production Association occupationally exposed to chronic low-dose-rate radiation have shown a significant increase in the incidence of ischemic heart disease (IHD) causally associated with the total external gamma-ray dose. The association remains after correction for multiple competing factors such as smoking and alcohol consumption.
Previous proteomics study using fresh-frozen tissue obtained at autopsy from the cardiac left ventricle from Mayak workers showed a dose-dependent downregulation of proteins involved in mitochondrial energy metabolism, cardiac cytoskeleton, and oxidative stress response when compared to the samples from non-irradiated controls. However, in this study, the individuals in the occupationally exposed group (>500 mGy) were on average 10 years older than the control subjects. We applied a statistical method to distinguish the dose-only dependent protein expression changes from those of the age-only dependent changes. The analysis indicated that the majority of the proteins were deregulated by the radiation dose and not the age . However, the confounding role of age could not be totally excluded. To address this issue, we initiated a new study where the control group was older than the irradiated group. Due to the difficulty of obtaining additional fresh-frozen material, we decided to use formalin-fixed, paraffin-embedded (FFPE) left ventricle tissue in the new study.
FFPE tissue is an ideal source of analytical material for clinical investigations due to its stability in long-term storage. The technical improvement of proteome analysis using FFPE material is a prerequisite for the successful elucidation of biological pathways. However, in spite of several technical improvements, proteomic analysis using FFPE material as an alternative to fresh-frozen tissue remains a challenge . The harsh conditions typically used for fixation have previously prohibited successful protein identification and reproducible protein quantification. During the formalin fixation proteins undergo degradation and cross-linking. The fixation results in a 30 Da Hydroxymethyl modification, tagged mainly on lysine residues contributing to the inter- and intramolecular cross-linking reactions. Lysine modification affects the efficiency of protein extraction and digestion. The Mass Spectrometry analysis often showed a general preferential detection of tryptic peptides with the C-terminal arginine over lysine indicating that modified lysine residues are inaccessible to protease during digestion.
A variety of methods that have been developed allowing the extraction of proteins from FFPE samples have proven to be quite successful in performing proteomics analysis. However, only a few proteomics studies using human FFPE cardiac tissue exist, probably due to the lack of this type of biomaterial.
The data presented here show the compatibility of FFPE tissue for a proteomic approach. The study suggests that chronic radiation exposure induces alterations in the heart metabolism, structure and oxidative stress response that may contribute to the radiation-induced IHD. Furthermore, it shows that these alterations are not caused by normal ageing.
Among the quantified proteins with two or more unique peptides, the expression level of 196 proteins was significantly different between irradiated samples and controls (2 unique peptides; ±1.3-fold; p < 0.05). Of these, 105 proteins were downregulated and 91 upregulated in the irradiated samples (Table 1).
Table 1. Significantly deregulated proteins in irradiated cardiac left ventricle proteome. The proteins listed in the table were identified and quantified by two or more unique peptides; fold changes and p-values are shown.
A volcano plot of all quantified and significantly deregulated proteins is shown in Figure 1A. The heat map based on the intensity of differentially regulated proteins in all individual samples showed a clear difference between the control and irradiated groups (Figure 1B).
Figure 1. Graphical representation of quantitative proteomics data of human FFPE cardiac tissue after chronic irradiation. Proteins are ranked in a volcano plot according to the −log10 of their statistical p-value (y-axis) and log2 fold change (x-axis). The green and red points represent the significantly downregulated and upregulated proteins, respectively (A). The heat map shows hierarchical clustering (average linkage, Spearman ranked correlation) of significantly deregulated proteins in 5 controls (Ctrl) and 15 irradiated (IR) samples (B). The green bars indicate downregulation and the red bars upregulation. The analysis was performed using Heatmapper web server.
Among the differentially regulated proteins were several lipid metabolism enzymes (Table 1). Long-chain-fatty-acid—CoA ligase 4 (ACSL4), hydroxyacyl-coenzyme A dehydrogenase (HADH), 2,4-dienoyl-CoA reductase (DECR1), enoyl-CoA hydratase (ECHS1), isovaleryl-CoA dehydrogenase (IVD), and enoyl-CoA delta isomerase 2 (ECI2) were all downregulated.
A number of proteins belonging to the sirtuin pathway and mitochondrial respiratory chain were also significantly downregulated in the irradiated samples (Table 1). The level of mitochondrial import inner membrane translocase (TIMM9), mitochondrial import receptor (TOMM22), and that of several proteins in the mitochondrial complexes I and V was reduced in the irradiated samples (Table 1).
Similarly, the data also showed downregulation in the expression of actin cytoskeleton components such as troponin I (TNNI3), troponin T (TNNT2), and different myosin isoforms including myosin heavy chain 14 (MYH14), myosin regulatory light chain 2 (MYL2), myosin light chain 4 (MYL4), and myomesin-1 (MYOM1) (Table 1). In contrast, several collagen isoforms were upregulated in the proteomics data, including collagen type XII alpha 1 chain (COL12A1), collagen type XV alpha 1 chain (COL15A), collagen type VI alpha 1 chain (COL6A) and collagen type VI alpha 2 chain (COL6A2) (Table 1).
The levels of several proteins of the oxidative stress response, namely catalase (CAT), peroxiredoxin 2 (PRDX2), peroxiredoxin 4 (PRDX4), extracellular superoxide dismutase [Cu-Zn] (SOD3), glutathione hydrolase 5 (GGT5), and glutaredoxin-1 (GLRX), were altered, most showing upregulation (Table 1).
A detailed analysis of the functional interactions and biological pathways of the deregulated proteins was performed using Ingenuity Pathway Analysis (IPA) software. Fatty acid metabolism, sirtuin signaling, mitochondrial dysfunction, protein ubiquitination, tissue fibrosis, cytoskeleton organization, and oxidative stress were the most affected pathways in the irradiated samples compared to the control (Figure 2A). Especially two biological networks showed enrichment of deregulated proteins. The predicted regulators of these networks were the peroxisome proliferator-activated receptor (PPAR) alpha and transforming growth factor beta-1 (TGFB1) (Figure 2B,C). PPAR alpha was predicted to be inactivated in irradiated samples, whilst TGF beta was predicted to be activated. Differentially expressed proteins were associated with cardiotoxicity-related pathways including cardiac dilation, cardiac enlargement, cardiac dysfunction, heart failure, and cardiac fibrosis.
Figure 2. Pathway analysis of differentially regulated proteins from human FFPE heart tissues. The most significant canonical pathways altered by irradiation are shown. The analyses were generated using Ingenuity Pathway Analysis (IPA) (QIAGEN Inc., Hilden, Germany). The bars indicate canonical pathways and the y-axis displays the −(log p) enrichment significance. The tall bars are more significant than the short ones (A). Graphical representation of the deregulated protein networks with their upstream transcriptional regulators peroxisome proliferator-activated receptor (PPAR) alpha (B) and transforming growth factor beta (TGFB) (C) are shown. The upregulated proteins are marked in red and the downregulated in green. The nodes in blue (inhibition) and orange (activation) represent transcription factors. The full protein names are given in Table 1.